260 Trop Anim Prod 1977 2:3

Present address Secretaria de Recursors hidraulicos, Comision de Rio Panuco, Tampico1

Technical Officer of FIRA, Banxico SA, attached to DIES2

In receipt of fellowship from IDRC, Canada6

Technical Officer of Banco de Credito Rural, Chetumal3

Present address Institute of Ecology and Genetics, University of Aarhus, Ny Munkegade, DK4

8000 Aarhus C, Denmark On leave of absence from University of NSW England, Armidale NSW 2351, Australia5

This unit has now been reestablished as the Deparamento de Investigación y Estudios7

Superios, Escuela de Medicina Veterinaria y Zootecnia, Universidad de Yucatan, Merida

RUMEN FUNCTION IN CATTLE GIVEN SUGAR CANE

R E Valdez , F J Alvarez , H M Ferreiro, F Guerra J Lopez , A Priego, T H1 2 6 , 6 3

Blackburn , R A Leng and T R Preston4 5

Centro de Investigación y Experimentacion Ganadera 7

Chetumal Q R Mexico

Diets based solely on sugar cane, urea and minerals support only maintenance in ruminants andprovision of supplements for production appears to be essential, As part of a study of the role of thesesupplements, a number of measurements of rumen function were examined in bulls given sugar canediets with or without rice polishings (a supplement which has given consistent responses in terms ofproduction). Determinations were made of pH, ammonia, total and VFA proportions and protozoanbiomass in rumen fluid on several days and for 28 hr consecutively. A new method of assessingprotozoan biomass is put forward. There were no apparent differences due to the rice polishings in any ofthe parameters of rumen function measured over 28 hr or between days. The outstanding findings werethat large holotrich protozoa populations existed in the rumen of these animals and that concentrationsvaried within and between days. The reasons for, and the effects of such fluctuations are discussed. It isconcluded that the value of rice polishings in sugar cane diets is not mediated through an effect on rumenfunction but is probably due to its ability to supply essential nutrients.

Key words: Sugar cane, cattle, rumen function, protozoa, rice polishings

The humid tropics have great potential for both crop and animal production,nevertheless there has not been the development of intensive beef and milk systemsas has occurred in the temperature regions. This lack of development appears to havebeen mainly due to a scarcity of cereal grain and to the fact that tropical crops aregenerally low in protein and are apparently of low nutritive value.

Recently systems of feeding sugar cane to ruminants have been developed (Boneferet al 1975, Leng & Preston 1976, Preston 1977); these would appear to haveconsiderable potential since dry matter yields from this crop are very high (oftenexceeding 40 tons/ha; Preston et al 1976 ) and the material is highly digestible(60-65%) (Montpellier & Preston 1977a, Ferreiro & Preston 1977).

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The technology for utilizing high levels of molasses in ruminant diets with consistentlyhigh rates of production is now well established (see Preston and Willis 1974).However, the utilization of whole sugar cane appears to be less predictable. Cattlegiven shredded whole sugar cane supplemented only with urea and minerals did notgrow; whereas, after addition of 1 kg/d of rice polishings, production rose to 0 9 kg/d(Preston et al 1976 ).

A dry matter digestibility in the range of 60-65% indicates that sugar cane should becapable of supporting higher rates of production than have so far been obtained.While, on a unit area basis, calculations suggest that up to 20 cattle could befattened/ha/year on this crop if the apparent limitations to its use could be overcome.

A puzzling feature of work reported recently using different supplements for sugarcane diets (Silvestre et al 1977), indicated that proteins of animal origin often appearto have no beneficial effects, and that vegetable protein supplements of which themost consistent has been rice polishings, are more effective (Preston et al 1976 ;Lopez et al 1976).

The objective of the present studies was to increase our understanding of digestiveprocesses in cattle fed sugar cane, with and without supple-. meets of rice polishings,in the belief that further knowledge in this area would lead to the development of morepredictable feeding systems with this crop. This initial investigation was aimed atexamining rumen function since it was expected that the rumen might be the effectivesite of action of the rice polishings.

Material and Methods

Experimental Animals and Diets: 8 crossbred (Brown Swiss/Zebu) bulls 18 months ofage and weighing between 300 and 400 kg were used in this study. They werehoused in single pens in an open-sided animal house. Mean day and nighttemperatures were 32 and 28! at the time the experiment was carried out. No artificiallight was used and day length was about 14 hr.

The animals were prepared with permanent rumen fistulas and were well accustomedto the experimental procedure. The diets consisted of freshly cut and shredded maturewhole sugar cane, approximately 12-14 mth old, 18 Brix (percentage sugar in juice)and 28% dry matter. The sugar cane was supplemented with a solution of urea inmolasses (817 g final molasses of 88 Brix, water 208 g and urea 283 g/litre) whichwas sprinkled on the surface of the sugar cane in the ratio of 50 ml/kg fresh cane. Theanimals had free access to a mineral mixture (50% NaCl, 45% ground rock phosphate(12% P) and 5% of a commercial trace element mixture (included Fe, Mg, Mn, Zn, Cu,Co). Four of the animals received in addition a supplement of 1 kg/d of rice polishings,the approximate composition of which was 26% starch, 13% crude protein, 13% lipids.

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The daily feeding regime was as follows: all the rice polishings (according totreatment) was given at 7:30 a.m. and the sugar cane/urea molasses mixture at 8:00a.m., the animals were fed again with the sugar cane/urea molasses at 4 p.m. Thesugar cane mixture was given in excess of requirement. The animals had beenestablished on the diet for 3 months before the experiment was initiated.

Experimental Procedures:Fibre digestion in the rumen: The nylon bag technique was used to measure thedigestibility of whole sugar cane and of fibre in the following way. Fifty g of freshlyshredded whole sugar cane were placed inside the bags which were then put into therumen of cattle which were being fed on the same diets. After 72 hr the bags wereremoved and washed 2 or 3 times by squeezing in water prior to drying at 100!for 24hr. The insoluble dry matter in 50 g samples of fresh sugar cane was estimated bywashing out the solubles, placing the bag in water and squeezing over a period ofabout 1 hr. These control bags were then dried and treated in the same way assamples from the rumen.

Sampling Rumen Fluid: Rumen fluid was sampled through a perforated copper tube (5cm long, 0.3 cm diameter) covered with nylon. This was attached to a flexible tube(0.5 cm diameter) which was passed through a stopper in the mouth of the rumencannula, allowing samples to be taken without opening the rumen cannula. Rumenfluid samples were taken from the animals at intervals over 17 days. On each daysamples were taken before feeding and at intervals for up to 6 hr after feeding. In oneperiod, samples were taken at hourly intervals over 28 hours. The sample of rumenfluid (20 to 50 ml) was taken with a minimum of suction, its pH and protozoa biomassdetermined immediately and samples (20 ml) stored at -15 after adjusting to pH 4 with10N H SO . 2 4

Analytical Methods:

Total VFA and proportions: Total volatile fatty acid (VFA) concentration in rumen fluidwas determined by titration after steam distillation. Proportions of VFA as acetic,propionic, and butyric acids were estimated by chromatography, after extraction intoether, using a Carle 311 gas chromatography (Carle Instruments inc., Fullerton,California) with a thermal conductivity detector and hydrogen as the carrier gas.Rumen fluid (4 ml) was placed in a small stoppered tube and 0.08 ml 50% (v/v) HSO2 4added followed by 2 ml ether. The tubes were stoppered and shaken by inverting 30times and placed in a deep freeze at -15 . Following freezing the ether was pouredinto a chilled test tube and about 50 mg anhydrous MgSO4 was added to remove anyresidual water. After standing for 5 min 2-5 ml were injected into a 2 m, 0.3 cmdiameter stainless steel column containing LAC-IR-296 (Burrell Corp. Pittsburgh, Pa).Proportions were calculated by reference to known standards prepared in the sameway (Holdeman and Moore 1972).

Ammonia in rumen fluid: This was estimated by a modification of the indoplenolmethod of Chaney & Marbach (1962). Rumen fluid (1 ml) was added to a 100 ml widemouth flask with a lid that was sealed by a plastic washer; a small hole was drilled inthe lid. A scintillation vial containing 0.5 ml 0.28N H SO was placed inside and the lid2 4tightly closed. One ml 5N NaOH was injected into the rumen fluid using a syringe and

Trop Anim Prod 1977 2:3 263

needle and the hole sealed with a piece of waxed paper. The flasks were allowed tostand at room temperature for 8 hr and then H O (5 ml) was added to the acid in the2scintillation vial and 1 ml was taken for analysis according to Chaney and Marbach(1962). Recoveries of ammonia from standard solutions were 80-90% and a standardcurve and blanks were always analysed in the same way and at the same time asrumen fluid.

Protozoan biomass: Microscopic examination showed that large protozoa were mainlyinvolved, which were difficult to count accurately. Large variations occurred betweendifferent counts in the same samples. Counting was also found to be too slow for thelarge number of samples involved. A Technique was therefore developed for rapidassessment of protozoan biomass based on a packed cell volume measured asfollows: Glucose (100 mg in 1 ml) was placed in a centrifuge tube and 15 ml of rumenfluid added with mixing. The tube was incubated at 40!for 20 min. Much of the solidmatter in rumen contents floated to the surface because of gas production and couldbe removed with a pasteur pipette. The protozoa gradually settled and after 20 minthe tubes were lightly centrifuged (500 r.p.m.) and the rumen fluid was removed toleave the protozoa in 1 ml. This was then mixed, transferred to a 1 ml graduatedhematocrit tube and incubated for 5 min at 40! Again l considerable separation ofprotozoa and residual feed materials was achieved in the tube. The tube werecentrifuged at 1500 r.p.m. for 1 min and the height of protozoal biomass wasimmediately read off the tube as a percentage of the volume. In the majority ofanalyses, particularly where protozoa were in large numbers, a clear demarcation wasobtained between protozoa and plant material. With small amounts of protozoanbiomass (less than 0.1% of rumen fluid) there was difficulty in separation. Repeatedanalyses on the same sampled indicated that individual biomass was estimated with acoefficient of variation of about ± 8% at a content of about 1% biomass (volume basis)in the rumen.

Results

Preliminary studies indicated that there were considerable fluctuations in protozoapopulation between animals and within animals on the same day. For this reasonparameters in rumen function have been monitored over a series of days and also ona continuous basis throughout a 28 hr period.

Digestibility of fibre: The digestibility coefficients obtained by the nylon bag techniquewere: 53.0 ± 1.3%(mean and SE of 8 estimations) for dry matter and 19.5 ± 2.2% (8determinations) for insoluble material.

Rumen fluid pH: Generally pH values were high over the full 24 hr period, howeverthere was a tendency towards a diurnal pattern characterized by a fall in rumen pHover a 6-8 period after feeding, with subsequent recovery to high values prior to thenext feed (figure 1). This trend was also apparent between days (figure 1). There wereno obvious differences in rum pH between animals with and without rice polishings.

Rumen fluid ammonia concentration: Rumen ammonia concentrations rose afterfeeding in both groups of animals but fell at about 4 hr after feeding and then slowlydeclined throughout the rest of the 24 hr period. In the case of the animals without rice

264 Trop Anim Prod 1977 2:3

Fig

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pH in

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Trop Anim Prod 1977 2:3 265

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266 Trop Anim Prod 1977 2:3

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Trop Anim Prod 1977 2:3 267

polishings, ammonia levels did not reach the same value after 24 hr as at the start ofthe study. However, ammonia concentrations in general were always very high relativeto many feeds (e.g. see Satter & Roffler 1977) (figure 2). This trend was alsoconsistent between days (figure 2).

Figure 4:Total VFA in rumen fluid during 28hr period (meanvalues and SE)( indicates feeding times)

Rumen fluid VFA proportions: The mean molar proportions of VFA over 2 hr areshown in figure 3. In general there was a decline in the proportion of acetate and arise in the proportion of propionate after feeding; subsequently this pattern wasreversed. This trend was particularly evident between days (figure 3) for acetate andpropionate while butyrate tended to remain constant.

Rumen fluid VFA concentration: Peak rumen VFA production was apparently reachedsome 1 to 2 hr after feeding with a gradual decline subsequently. There were noobvious differences due to the rice polishings supplement. Mean values for the 28hrsampling period are given in figure 4.

268 Trop Anim Prod 1977 2:3

Fig

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5:P

roto

zoan

bio

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s in

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en fl

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(mea

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Trop Anim Prod 1977 2:3 269

Protozoan biomass in rumen fluid: Protozoa were present in the cattle on these dietsin large numbers. The predominant species were Isotricha intestinalis and Dasytrichasp; Entodinea sp were present at levels of less than 1 x 105/ml and were small inbiomass relative to the holotrichs. The method for assessment of protozoan biomassincluded the entodinea but was made up of at least 95% of the large holotrichs. Thediurnal changes in protozoan biomass were similar in all animals on both diets (figure5). Protozoan biomass rose rapidly after feeding, remained high for 6-8 hr and thenapparently slowly decreased to low or negligible levels immediately prior to themorning feed. During the 28 hr experiment the maximum protozoan biomass washigher in the animals on the diet with rice polishings (figure 5). However, this findingwas not supported by subsequent examinations of protozoan biomass on differentdays ( figure 5).

Discussion

The digestibility of the dry matter of mature sugar cane is in the range 60 to 64%(Montpellier & Preston 1977a; Ferreiro 8 Preston 1977), Sir the total content of solublesugars in mature cane is over 50% (See Ferreiro et al 1977), these finding indicatethat very little of the insoluble components is digested. This is corroborated by theresults of the nylon bag technique which indicate that the digestibility of the fibre wasonly 19%.

Effects of rice polishings: A considerable amount of work has been reported on theeffect of rice polishings in diets based on sugar cane and urea. The consistent resultsare as follow: no effect on digestibility of dry matter (Montpellier & Preston 1977b);significant positive linear effects ( for up to 1 kg/d of rice polishings) on voluntary feedintake, daily weight gain and feed conversion (Lopez et al 1976; Preston et al 1976 ).In contrast with these effects on the animal, the results of the present study showed:no systematic effect of rice polishings on any of the parameters of rumen functionmeasured here.

Overall rumen function: The outstanding findings in these studies were the large andvariable protozoa populations in the rumen of tattle given sugar cane diets. Thesewere largely the holotrich species (about 80% Isotricha and 20% Dasytricha). Otherspecies of protozoa which were present were a. insignificant proportion of the totalbiomass. On most cattle diets protozoa are generally present in the rumen but rarely insuch concentrations. Only on fresh pasture grass (Clarke 1965) or on grain basedrations fed in restricted amounts (Eadie & Mann 1970) are protozoa found in suchlarge numbers.

The technique developed to measure protozoa biomass has many advantages overconventional counting procedures, but its main virtue is the relative speed ofdetermination. Conventional counting procedures for the large protozoa are inherentlyinaccurate, however, at high protozoa biomass (greater than 1%) this new methodappears to be accurate to ± 8%. The procedure is put forward for comparativepurpose rather than to indicate total protozoa present in the rumen at any one time.This latter measure appears only to be possible in slaughtered animals where rumenfluid can be thoroughly mixed ( Minor et al 1977b).

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The pattern of change of protozoa biomass over a 24 hr period indicates that theprotozoa probably settle in the rumen on becoming full of starch as they do in testtubes. In studies of protozoan metabolism (Valdez, Preston & Leng 1976 unpublishedobservations) it was found that protozoa incubated in boiled rumen fluid withoutsubstrate rapidly clumped on the sides of the incubating flasks or attached themselvesto any fibrous material in the flask; whereas with additions of glucose, sucrose orglucose and sucrose, no clumping occurred even though the protozoa settled in theflasks if they were not shaken. In the flasks without substrate, clumps were observedto form within 30 seconds of cessation of shaking and there was a definite migrationto a clump. Microscopic examination of these clumps showed that there was noapparent adhesion of the organisms which were moving freely, suggesting that theword clumping is a misnomer and flocking may better describe this phenomenon. Thein vitro results tend to suggest that in the presence of substrate these organisms areactive. In vivo studies suggest that subsequently they become heavy and tend to sinkin the rumen and as soon as the substrate is exhausted begin to migrate into groups.In the feeding situations reported here, where the energy source is already in solution,the protozoa may confer an advantage to the host animal, in that they can rapidlystore soluble sugars which presumably are then fermented at a rate to meet theirrequirements for maintenance and reproduction, allowing the host animal a morecontinuous supply of VFA. The protozoa probably settle in the rumen once they havestored sufficient starch. On this same feeding regime it was reported that thefrequency of rumen contractions, and their strength increased after feeding (Priego &Leng 1976) which would tend to redistribute the protozoa through rumen contents at atime when substrate was readily available.

The migration and flocking and also the settling in the rumen would tend to reduce theoutflow of protozoa from the rumen (see Weller & Pilgrim 1974). In fact, subsequentstudies have shown that the concentration of protozoa in the omasum is negligibleeven though in the same animal concentrations in the rumen often exceeded 5% ofrumen volume as biomass (Minor et al 1977a). The increase in the apparentprotozoan biomass in the rumen shortly after feeding is probably due mainly to aredistribution of protozoa. Warner (1965) has observed that the holotrich protozoatend to be dividing just before feeding, and the differences in magnitude of theprotozoan biomass between days in the present study suggest that protozoa candouble their populations in 24 hr and also that there are some deaths of protozoa. Ifprotozoa die then the soluble parts of their cells presumably become substrates forthe remaining microbial community (see Leng 1976).

In the first experiment there was an apparent significant reduction in protozoanbiomass in cattle fed with rice polishings, however when the protozoan biomass wasexamined with time the between day variation was large and also there appeared tobe cyclical variation in the protozoan biomass. Overall, there appeared to be nodifferences between the groups of animals, supplemented with or without ricepolishings, the difference in the first 24 h sampling period being fortuitous.

Trop Anim Prod 1977 2:3 271

A point that should be emphasized is that in making comparisons of protozoalbiomass between days, only the maximum measurements of packed cell volume ofprotozoa should be used. This is still a minimum value for the protozoan biomasssince other studies from these laboratories have shown that point samples taken inthis way consistently underestimate the true population density when these aremeasured in mixed rumen fluid from slaughtered animals (Minor et al 1977b). Thevalues reported here are therefore to be considered as relative values only.

The overall results suggest that there are distinct phases in substrate utilization.Shortly after feeding, the protozoa are apparently actively storing starch from solublesugars and presumably bacteria are fermenting soluble sugars quite rapidly. Thedecrease in acetate and the increase in propionate shortly after feeding is thereforeprobably related to bacterial fermentation of soluble sugars. The large holotrichprotozoa are thought to produce acetic, butyric and lactic acids (Hungate 1966), andtherefore the second phase in which acetate rises to original levels before feeding isprobably largely due to fermentation of stored starch by protozoa with secondaryfermentation of lactic acid by bacteria to a mixture of VFA. With depletion of sugar insolution and of stored starch in protozoa, cellulose digestion which is minimal,increases in importance as indicated by higher acetate proportions with time. Thisconcept implies that a succession of microbial communities are developing andbecoming of increasing importance. Possibly all three processes can occursimultaneously provided the availability of rumen ammonia is high. Ammonia levelswere extremely high throughout the 24 h period examined and were in excess of thatrequired for instance for maximum starch utilization (see 0rskov 1976).

The main points brought out by this study are that there appear to be no differences inprotozoan biomass or any other parameter in the rumen of cattle given sugar canediets with or without rice polishings. The high values of protozoan biomass appear tobe maintained by high and constant pH values. Since little change occurs in rumenfermentation, this suggests that the effect of rice polishings on animal performance ispossibly mediated directly through provision of essential nutrients for absorption.

Acknowledgements

We wish to acknowledge the following for supporting this project: The Australian MeatResearch Committee and the International Development search Centre, Canada

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a sugar cane diet: effect of level of rice polishings and of processing the sugar cane byderinding or chopping Trop Anim Prod 1: 150-161

Preston T R 1977 Nutritive value of sugar cane for ruminants Trop Anim Prod 2: 125-142Priego A & Leng R A 1976 Ruminal Contractions in Cattle Fed Sugar Cane Trop Anim ProdSatter L D & Roffler R E 1977 Protein requirement and non protein nitrogen utilization Trop

Anim Prod 2: 238-259Silvestre R, MacLeod N A Preston T R 1977 Supplementation of sugar cane/urea for growing

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Received 7 November 1977